DE69615163T2 - TILE ADHESIVE WOUND SEALANT - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the invention

This invention relates generally to wound sealants and, more particularly, to a platelet adhesive wound sealant that can enhance wound healing.

State of the art

Repair of damaged tissues and vessels is a universal concern of all surgical specialties. Tissue damage, often as a result of surgical intervention, can be difficult to repair. Continued extravasation of fluid from or into a wound can increase a patient's morbidity, prolong the course of disease, and ruin an otherwise promising outcome.

Following trauma or surgery, the normal healing process is diminished, a difficulty that is exacerbated by the presence of concurrent diseases, such as diabetes, that further impede healing progress. Serum fluid exudate from tissue cut surfaces can slow wound healing and serves as a culture medium for infection. Fluid leaks occur in many tissues regardless of how precise the surgical technique. Vascular tissues continue to bleed, even through the eschar from electrocautery, and continue to produce exudate for long periods after blood loss has ceased.

Grafts in the arterial vascular tree, whether biological or synthetic, often leak at the suture line or through the graft material itself.

Pre-existing or medically induced coagulopathy often exacerbates this process. Wounds to the dura mater are inherently difficult to repair, and historical approaches have documented failure rates with persistent cerebrospinal fluid leaks of up to 35% for some types of procedures. Persistent air leaks from resected lung tissue are difficult to repair and can result in significantly prolonged recovery.

A variety of approaches have been taken to address these problems. Although widely used, topical hemostatic agents made from synthetic or highly modified biological materials have limited efficacy as sealants, are ineffective as binders, and result in inflammation against foreign material, which itself can prolong the healing process.

Fibrin glue, formed by cleaving fibrinogen with thrombin and polymerizing the resulting fibrin, has been widely proposed as a general solution to these problems, and has even been called the perfect surgical sealant. Countless attempts have been made to provide the surgeon with fibrin glue on demand. Fractionation of concentrated fibrinogen along with other clotting proteins from plasma by cold precipitation and density separation has been widely studied. Cryoprecipitation selectively extracts the components of the coagulation cascade while separating out the majority of plasma protein and the bulk of the fluid volume. The typical yield from 450 ml of blood is 10 to 12 ml.

Although widely used in Europe, commercially prepared cryoprecipitate pooled from donors as a substrate for fibrin glue was not available in the United States for many years due to regulatory concerns about potential contamination and disease transmission. Cryoprecipitate from a single donor is not free from the risk of disease transmission, and HIV infection following topical application of fibrin glue from cryoprecipitate pooled from a single donor donor was documented. Upfront donation of autologous blood or plasma for the production of autologous cryoprecipitate not only anticipates the need but also requires an outpatient, motivated patient who meets the strict donor criteria of blood banks, together with the geographic availability of a qualified processing facility.

Given these difficulties, numerous approaches have been developed to facilitate the availability of single donor and autologous fibrinogen concentrates. Although the results of these efforts have proven beneficial in a wide range of surgical applications, they all suffer from the disadvantages of complicated or lengthy processing techniques, high costs, the requirement for upfront donation, or a combination of such limitations. In addition, tissue adhesive sealants that rely primarily on fibrinogen for their function have been shown to hinder healing rates due to the high fibrinogen content required for their effectiveness. However, no inhibition of wound healing has been observed at fibrinogen concentrations less than 15 mg/ml, but rather have been shown to promote the early stages of wound healing.

Recently, there have been attempts to use autologous plasma, separated from whole blood by density differential rather than by gravity or centrifugation, as the substrate for a surgical topical tissue sealant after activation of entrained fibrinogen by thrombin in the presence of calcium ions. These methods result in a plasma product containing natural fibrinogen concentrations, which have been shown to be insufficient on their own to reliably form a suitable fibrin gel. The strength of fibrin gels is directly related to fibrinogen concentration, and naturally occurring concentrations are at best at the lower limit for forming tissue sealants and adhesives. Due to illness, dilution by the anticoagulant solution required for blood collection, blood thinning coinciding with the surgical procedure itself, or a combination of such factors, subnormal concentrations are often encountered, resulting in fibrin gels with unacceptable mechanical and adhesive properties.

All fibrinogen-based sealant adhesives depend for their function on that part of the coagulation cascade known as the common pathway, through the conversion of fibrinogen to fibrin monomer and then its polymerization and cross-linking to form a stable network. Initiation of this process is usually accomplished by admixing commercially obtained concentrated thrombin in aqueous solution containing calcium ions. It is generally held that in order to obtain a suitably functional and effective fibrin sealant adhesive, it is necessary to include a small concentration of dissolved calcium salts in the thrombin activator to provide calcium ions. The concentration of dissolved calcium required varies from as little as 0.0036 to as much as 0.072 mg Ca++ per unit of thrombin. Addition of calcium ions to thrombin results in increased costs, additional manufacturing steps, and increased complexity.

However, the presence of calcium ions together with platelets in a clot enabled the activated platelets to contract and expel serum fluid from the clot, thereby significantly reducing its size and reducing its adhesive and sealing properties. Platelets were thus rigorously removed from most fibrin glue preparations, or if present, their concentration was generally limited to a level below physiological. The higher the platelet concentration, the higher the likelihood that the addition of calcium ions would result in undesirable shrinkage of the clot.

However, increased platelet concentrations have been shown to be valuable in accelerating wound healing. Platelet-based wound healing extracts are decoctions of defibrinated plasma that lack clotting ability. Use of these preparations requires the addition of a carrier, such as microcrystalline collagen, to increase viscosity and provide sufficient mechanical integrity for targeted application. These carriers may themselves induce an inflammatory response against foreign material, thus inhibiting or preventing any wound healing benefits.

It can therefore be recognized that known methods and techniques for improving wound sealing and promoting wound healing do not meet the expectations of providing a reliable, biocompatible, therapeutically effective composition. Foreign materials induce inflammatory reactions against foreign material which delay or inhibit healing. Homologous products carry the risk of disease transmission or bioincompatibility reactions. Highly concentrated fibrinogen solutions are time-consuming to prepare and inherently inhibit wound healing. Whole plasma substrates, including those containing platelets as enhancers, often have fibrinogen concentrations too low for effective use and rarely contain platelets at concentrations considered useful for accelerating wound healing. Extracts of platelet concentrates, although considered effective for accelerating wound healing, are not capable of acting as wound sealants or adhesives by themselves.

Accordingly, there remains a need for an effective wound sealant that can be prepared safely, easily and quickly, and that preferentially enhances or accelerates the healing process.

SUMMARY OF THE INVENTION

The present invention provides a plasma-buffy coat concentrate comprising plasma, platelets at a concentration of at least 1.0 x 109 cells/ml, and fibrinogen at a concentration of at least 5 mg/ml. When the plasma-buffy coat concentrate is combined with a fibrinogen activator at a concentration sufficient to initiate the formation of a clot, a platelet adhesive wound sealant is formed. In a preferred embodiment, the wound sealant contains white blood cells at a concentration of at least 3.0 x 107 cells/ml and can be used to reduce or treat infection and, in conjunction with platelets, to promote wound healing.

A method of processing blood to produce the plasma buffy coat concentrate is also provided. The method involves centrifuging anticoagulated blood to remove red blood cells and produce a plasma buffy coat concentrate. To produce the plasma buffy coat concentrate, water is removed from the mixture. As described above, a fibrinogen activator is mixed with the plasma buffy coat concentrate to produce a wound sealant which can then be applied to a wound to facilitate wound sealing and healing.

This method of manufacturing a wound sealant is performed perioperatively in a simple manner using the patient's own blood. The method and compositions of this invention are generally suitable for mammals and find particular use in humans.

DETAILED DESCRIPTION

The present invention provides a plasma-buffy coat concentrate containing plasma and concentrated platelets and fibrinogen. In a preferred embodiment, the plasma-buffy coat concentrate also contains concentrated white blood cells. The plasma-buffy coat concentrate can be combined with a fibrinogen activator to produce a wound sealant.

The present invention also provides a method of processing blood which produces a plasma-buffy coat concentrate of the invention and which can be used to prepare a platelet adhesive wound sealant which can promote wound sealing and wound healing. The method comprises centrifuging anticoagulated blood to remove red blood cells and produce a plasma-buffy coat mixture. Water is removed from the plasma-buffy coat mixture to produce a plasma-buffy coat concentrate. The concentrate is mixed with a fibrinogen activator to produce a Wound sealant is produced that seals the wound upon contact with damaged tissue and can promote wound healing. The procedure can be performed perioperatively to produce a platelet adhesive wound sealant from autologous blood.

The plasma buffy coat concentrate and platelet adhesive wound sealant of the present invention are described in detail below. Methods for preparing the compositions of the present invention are then described in detail herein.

The plasma buffy coat concentrate/platelet adhesive wound sealant

The plasma-buffy coat concentrate of the invention comprises plasma containing platelets in a concentration of at least 1.0 x 109 cells/ml and fibrinogen in a concentration of at least 5 mg/ml. Preferably, the platelet concentration is from 1.0 x 109 cells/ml to 2.5 x 109 cells/ml. In a preferred embodiment, the fibrinogen concentration is from 5 to 15 mg/ml.

The plasma buffy coat concentrate may additionally contain white blood cells, preferably at a concentration of at least 3.0 x 107 cells/ml. More preferably, the concentration of white blood cells is from 3.0 x 107 cells/ml to 6.0 x 107 cells/ml. In a preferred embodiment, the white blood cells present in the plasma buffy coat concentrate comprise 60 to 70% lymphocytes, 15 to 25% monocytes and 5 to 25% neutrophils.

A platelet adhesive wound sealant according to the invention comprises the plasma buffy coat concentrate described above and a fibrinogen activator in a concentration sufficient to initiate clot formation. Fibrinogen activators are well known and include, for example, thrombin and batroxobin. The fibrinogen activator may be present in various concentrations depending on the desired time period for clot formation. When the fibrinogen activator is thrombin, at thrombin concentrations greater than 100 units per ml or thereabouts in wound sealant, fibrinogen concentration becomes the rate-limiting step of clotting. Higher thrombin concentrations can be used, but no additional benefit is obtained. Furthermore, there is a greater risk of thrombin leaking into the vessel interior. At concentrations below about 100 U/ml, thrombin concentration is the rate-limiting substance in the wound sealant. Thus, thrombin concentration can be used to control the time to gel formation. The relationship between thrombin concentration and clotting time is discussed in the Methods section below.

Although fibrinogen activator is generally added exogenously, thrombin in plasma-buffy coat concentrate can be generated from endogenous prothrombin present in the plasma component of plasma-buffy coat concentrate. The anticoagulant citrate in the concentrate binds calcium ions required to facilitate the cleavage of prothrombin and generation of thrombin in the coagulation cascade. Therefore, to generate thrombin from prothrombin present in plasma-buffy coat concentrate, calcium is added in an amount sufficient to facilitate the progression of the coagulation cascade.

Calcium salts such as about 10% calcium chloride, which is commercially available as a sterile pharmaceutical solution, are conveniently used. The amount of calcium salt required to recalcify the plasma buffy coat concentrate sufficiently to facilitate the generation of endogenous thrombin for clot formation depends on the residual amount of citrate anticoagulant present in the preparation. However, the addition of one part 10% calcium citrate or equivalent calcium from another source to 11 to 12 parts plasma buffy coat concentrate is sufficient. The use of calcium to activate thrombin in the plasma buffy coat concentrate is advantageous in procedures where a clotting time of one to two minutes is desired. Such clot formation periods may be useful in bone defect procedures where bone or a bone growth matrix is added to the preparation prior to clot gelation.

The blood donor for the platelet adhesive wound sealant of the invention is preferably of the same species as the damaged tissue to which the wound sealant is applied. The wound sealant is generally suitable for mammals, but is preferably used for humans. Preferably, the blood types of donor and recipient are matched, as in standard blood donation practices for the respective species. If possible, the blood donor is preferably the same animal to which the wound sealant is applied. If possible, the wound sealant is preferably prepared either perioperatively or immediately before use.

The fibrin clotting process and the function of platelets are common to all mammalian species. However, it is clear that the closer the relationship between the donor and recipient of the platelet adhesive wound sealant of the invention, the better it is for the recipient in terms of the risk-benefit ratio and the likelihood of a desired result. The wound sealant of the invention is particularly suitable for self-administration since it can provide significant volumes of an effective agent in less than thirty minutes, thus avoiding the risks and complications associated with allogeneic blood transfusions.

Using the Platelet Adhesive Wound Sealant

Although the wound sealant composition of the invention is particularly suitable for use in humans, it is generally suitable for mammalian species. For example, in horses, leg wounds, particularly full-thickness skin abrasions, are in principle difficult to heal. The application of the platelet-containing wound sealant accelerates the healing of wounds of this type compared to conventional treatments.

The use of foreign material may be unavoidable if the recipient’s blood volume is not even sufficient for a temporary collection of the blood required for processing into a plasma-leukocyte cuff mixture. blood volume. For example, in pediatric cardiac surgery, the use of fibrinogen sealants has been recognized as very valuable, but the children themselves are too small to provide their own blood for processing. However, the volume required of such prior art sealants often exceeded that which can be obtained from a single donor, greatly increasing the risk of complications. However, by using the wound sealant of the present invention, a single unit of ordered whole donor blood can be processed for use as a wound sealant. While the red blood cells are used to prime the pump and the plasma is used for postoperative correction of clotting factors, the plasma-buffy coat mixture can be used to prepare an effective topical adhesive sealant, significantly reducing postoperative blood loss without increasing the number of donor exposures.

In addition to those listed above, a wide range of beneficial human applications have been studied and documented. A number of self-applications have been performed with a high success rate and no complications. The platelet adhesive wound sealant of the invention has been used to seal the leakage of cerebrospinal fluid through incised dura mater; to seal anastomoses of natural vascular grafts and vascular prosthetic grafts; in surgeries involving significant incisions such as radical prostatectomy, tram flap reconstruction surgery, radical neck surgery, etc.; in plastic surgery including burn transplants and other free skin transplant applications; and in vascular-rich incised tissue such as kidneys, liver and spleen. The wound sealant of the present invention was uniformly effective to eliminate or greatly reduce postoperative bleeding and extravasation or loss of serum or other fluid in these applications.

It has been shown that the wound sealant of the invention, mixed with bone meal, provides rapid and uniform bone regrowth in craniofacial and orthopedic procedures. Healing was also diabetic non-healing wounds, achieving successful wound closure where weeks of conventional therapy had failed. The wound sealant of the invention was also successful in healing septic wounds with long-standing resistance to standard approaches, including antibiotic-resistant bacterial infections.

When the wound sealant of the invention was applied to the sinus cavities after endogenous sinus surgery, it was observed that regrowth of mucous membrane was more rapid and uniform than with conventional treatment methods. Inner ear surgery was also successful, with bone prostheses from the cochlea successfully attached to the eardrum, and also for reconstruction of the eardrum itself. A few milliliters of wound sealant were allowed to gel in a medicine cup, transferred to an absorbent pad, and compressed to secrete serum and form a thin compress of fibrin, platelets, and white blood cells. This compressed clot was then dried under a heat lamp for 30 minutes, forming a dry, firm but flexible layer. This layer was then cut to the correct size, sewn in place of the missing eardrum with a few fine resorbable sutures, and packed externally and internally in platelet adhesive wound sealant. Restoration of a functioning eardrum was observed within six weeks, with resorption and disappearance of the wound sealant according to the invention.

The platelet adhesive wound sealant has been used clinically in the repair of drill holes (bone milling holes) in the skull by mixing plasma buffy coat concentrate with autologous bone mass from the drilling procedure as the bone growth matrix. The platelet adhesive wound sealant has also been used together with autologous bone graft (iliac crest), autologous bone chips, cadaveric bone, and demineralized bone matrix in the repair of bone defects of the spine. The platelet adhesive wound sealant has also been used together with autologous bone graft (iliac crest and rib) in the repair of poorly healing pathological mandibular fracture. In one mandibular repair case, a string of antibiotic-impregnated methyl methacrylate beads was incorporated into the wound sealant, embedded in the soft tissue outside the mandibular bone graft, and encapsulated with additional platelet adhesive wound sealant. When using the platelet adhesive wound sealant in bone defect applications, physician evaluation of bone ingrowth was always good to excellent. All grafts ingrowthed and there were no associated pathological conditions. Other sources of bone growth matrix such as hydroxyapatite or bone marrow may also be used in conjunction with the wound sealant of the present invention.

Other uses of the platelet adhesive wound sealant will be apparent to those skilled in the art, including the addition of drugs or other chemicals for localized, controlled application.

Production of anticoagulated whole blood

Whole mammalian blood is collected by aseptic or sterile technique from any accessible site. The blood is anticoagulated at the time of collection by use of a citrate-based anticoagulant. Any citrate-based anticoagulant is suitable. Standard donor blood collection bags contain citrate-based anticoagulants. For example, those manufactured by Terumo Corporation (Teruflex, CPDA-1) contain 63 mL of citrate-phosphate-dextrose-adenine anticoagulant to collect 450 mL of blood. Sixty-three milliliters of anticoagulant contains 206 mg each of citric acid (hydrous) USP, 1.66 g sodium citrate (hydrous) USP, 140 mg sodium dihydrogen phosphate (hydrous) USP, 1.83 g dextrose (anhydrous) USP, and 17.3 g adenine. Anticoagulant-treated blood can be stored at room temperature for up to five days. Preferably, anticoagulant-treated blood is stored for three days, more preferably 24 hours or less, because after this time platelet function begins to decline and clotting factors begin to degrade.

Alternatively, anticoagulation treatment can be performed using a double-tube blood collection set containing a citrate-based anticoagulant. Examples of suitable blood collection sets include Electromedics Model BT727BP and Haemonetics Model 247. Suitable citrate-based anticoagulants include that of Electromedics (Model ELMD-CPD). The anticoagulant contains 3 mg of citric acid (anhydrous) USP, 26.3 mg of sodium citrate (hydrous) USP, sodium dihydrogen phosphate (monohydrate) USP, and 23.2 mg of dextrose USP per milliliter. These blood collection sets facilitate mixing of a citrate-based anticoagulant at the same time as the blood is drawn. The amount of anticoagulant is not critical as long as there is enough anticoagulant to prevent clot formation. Generally, a ratio of 10 to 15 ml of the above anticoagulants per 100 ml of blood drawn is used. Higher concentration anticoagulant solutions may also be used, with appropriate adjustment of the administration ratio. Blood drawn by these sets is conveniently processed at the time of collection.

The blood is preferably collected at or near room temperature, anticated and stored if desired. The remainder of the procedure is also conveniently performed at or near room temperature. Temperatures below 18ºC or above 21ºC cause a more rapid decline in platelet function and degradation of desired clotting factors.

Preparation of a plasma-buffy coat mixture

Anticoagulated blood is then subjected to a centrifugal separation process to produce a plasma-buffy coat mixture containing the plasma-buffy coat components of whole blood in the plasma and removing red blood cells.

Such double centrifugation techniques for removing red blood cells are well known and devices for performing the separation are commercially available. Devices from several companies are suitable, such as Electromedics AT500, AT750, AT750EF, AT1000 and ELMD500; Dideco-Sorin BT795 series, STAT and STAT-P; Haemonetics Cell Saver 5; and Cobe-Gambro BRAT 2. The device is equipped with a standard blood centrifugal fractionation kit, which is a single-use kit and includes a centrifuge basket, tubing and collection bags for the procedure, and is available from the device manufacturers. Collection bags are connected to the outlet tubing of the centrifuge basket and blood supply tubing is connected to the inlet tubing of the processing set. Suitable sterile supplies for supply and collection are also commercially available, for example from Electromedics (BT727BP or BT727SP), Haemonetics (#247) and Cobe-Gambro.

The machine centrifuge is then preferably set to the maximum rotation speed. Although lower centrifugation speeds can be used, the highest rotation speed available allows for maximum yield with the fastest blood flows, thereby reducing processing times. In addition, using the highest centrifugation speed provides the highest packing density of red blood cells and the highest yield of platelets and plasma. Typical high-speed centrifuge speeds are 4800 to 5600 rpm, which produces centrifugal forces on the order of 2000 G. Blood flow is then started at flow rates of about 50 ml to about 150 ml per minute. Lower flow rates increase the time of the procedure without significantly increasing the yield. Higher flow rates do not allow separation of platelets from plasma, thereby lowering the yield. A flow of about 100 ml/minute at this stage provides balanced desired effects.

As blood flows through the centrifuge basket, the denser particles are pushed to the periphery while the less dense cell-free plasma is pushed to the center where it overflows into a collection bag. This process is preferably continued until the centrifuge basket is nearly full of particles and only a small central core of clear plasma remains. The cell pack typically consists of 70% to 80% particles and 20% to 30% plasma.

The blood pump is then stopped and the centrifugation speed is reduced to about 2400 rpm. With the now lower forces, on the order of about 250 G, the denser red blood cells and neutrophils remain at the periphery of the centrifuge basket, while the less dense platelets, monocytes and lymphocytes, the so-called "buffy coat", are pushed into the central plasma column, which becomes turbid. Lower rotation speeds may exert insufficient force to maintain the separation between red blood cells and plasma, which would prevent the collection of a buffy coat free of red blood cells.

The outlet tube leading to the plasma collection bag is then closed. The outlet tube leading to another collection bag is opened and blood flow is restored, at about 25 to about 125 ml/minute. Lower flow rates unnecessarily prolong the procedure, while higher flow rates reduce the yield of platelets. Blood now flowing into the centrifuge basket is again fractionated. At these lower centrifugal forces, the red blood cells and neutrophils remain in the centrifuge basket, while the plasma fraction continues to exit the basket, taking the majority of platelets, monocytes and lymphocytes with it into the second collection bag.

Continued pumping of blood into the basket so that it fills with red blood cells forces the central plasma column and entrained debris into the outlet tube. Blood flow continues until red blood cells begin to flow into the second collection bag, at which point blood flow is stopped. The volume of blood required to reach this point is inversely proportional to its hematocrit.

The outlet tube to the platelet bag is then clamped and the centrifuge stopped. The plasma collection bag is then opened. The direction of the blood pump is reversed and the platelet-poor red blood cells are passed into a third collection bag. If necessary, some or all of the cell-poor plasma can be recombined with the red blood cells. The packed red blood cells, the cell-poor plasma, or the recombined Platelet-poor whole blood can be returned to the patient immediately, retained for later reinfusion, or discarded. A single cycle of filling, fractionating, and emptying takes approximately 10 to 13 minutes, depending on the capacity of the centrifuge basket, the hematocrit of the blood collected, and the flow rates selected. The procedure can be repeated to provide larger volumes of blood component fractions as desired.

The volume yield of the various fractions depends on the same parameters that affect processing time. Typically, a 125 mL centrifuge basket yields about 125 mL of packed red blood cells, about 100 to 350 mL of cell-poor plasma, and about 25 to 60 mL of buffy coat, depending on the hematocrit of the blood processed. Larger centrifuge baskets yield proportionally larger volumes. Successively larger volumes of blood collected place a greater burden on the donor and may dictate the size of the centrifuge basket used and the speed or total volume of processing.

The yield of platelets in the buffy coat is similarly dependent on the parameters affecting cycle time and volume yield, as well as the number of platelets in the blood collected and the relative densities of the constituents. The yield ranges from about 50% to about 80% of the platelets entering the basket, with about 60% to about 70% being the norm. Platelet concentrations in this buffy coat fraction range from about 350,000,000 to about 800,000,000 per ml, with about 500,000,000 to about 650,000,000 being the norm. Fibrinogen concentration is at or below that of the donor blood.

After processing, the plasma-buffy coat mixture can be stored in appropriate platelet storage containers for periods of up to 5 days at room temperature, but platelets perform best when used within 36 hours of collection. If platelets are stored for more than 24 hours after the time of collection, they require continuous agitation.

Preparation of a plasma-buffy coat concentrate

Water is removed from the plasma-buffy coat mixture to produce a plasma-buffy coat concentrate. This step is conveniently performed using a conventional hemoconcentration device.

Commonly available blood bag spikes, stopcocks and tubing are assembled so that the collection bag containing the plasma-buffy coat mixture is connected to a three-way stopcock. A fluid transfer set with a female luer lock (Codon Medlon Inc., catalog number B310), comprising small diameter PVC tubing equipped with a clamp lock and fitted with a blood spike at one end and a female luer lock at the other end, is connected to the plasma-buffy coat mixture collection bag via the blood spike. A three-way stopcock (commercially available from a number of sources including Medex, Inc.) is attached to the female luer lock of the transfer set. A 60 cc syringe (commercially available from a number of sources, including Pharmaseal, Inc.) is attached to one arm of the stopcock. The third arm of the stopcock is fitted with a male luer lock adapter (commercially available from sources such as Minntech Corp.) to 1/4" tubing, which in turn is connected to a piece of 1/4" PVC tubing (approximately 2 to 3 cm long).

A hemoconcentrator (commercially available from a number of sources including Minntech Corp., Amicon Corp. and others) is then connected to the 1/4" tubing at its inlet. The outlet of the hemoconcentrator is also connected to a piece of 1/4" PVC tubing (about 2 to about 3 cm long) which serves as the attachment point for the inlet coupler of a blood transfer bag (commercially available from Terumo Corporation and other sources). The plasma-buffy coat mixture can then be drawn into the syringe. By adjusting the stopcock, the flow path between the syringe and the hemoconcentrator is opened. At the discharge port of the A vacuum of about -400 torr is then applied to the hemoconcentrator and the syringe plunger is actuated to force the plasma-buffy coat mixture through the blood path of the hemoconcentrator at 20 to 35 ml/min. A vacuum of about -100 to about -400 torr may be used to draw water from the plasma-buffy coat mixture to form a plasma-buffy coat concentrate, with the flow rate of the mixture adjusted inversely proportionally. Once all of the plasma-buffy coat mixture has flowed into the hemoconcentrator, the vacuum is released. The empty syringe is replaced with a syringe filled with a physiological solution substantially free of calcium ions. Physiological saline, citrated plasma, phosphate buffered saline and low-salt albumin are suitable. A 20 cc syringe is conveniently used. The physiological solution is introduced into the hemoconcentration device to flush residual platelets and plasma-buffy coat concentrate from the hemoconcentration device into a receiving transfer bag.

The amount of time required to remove water from the plasma-buffy coat mixture to produce a plasma-buffy coat concentrate varies depending on the flow rate and the volume processed. The final volume of the plasma-buffy coat concentrate varies as a function of the relationship of flow rate to vacuum level. Ideally, the volume of the plasma-buffy coat mixture is reduced by about 50% to about 66%. Reductions of about 25% to about 50% are suitable, but not preferred. Reductions of more than about 70% carry the risk of producing a liquid with such high viscosity that flow may be difficult or impossible.

This hemoconcentration step in the procedure is best carried out at room temperature.

The plasma-buffy coat concentrate is a viscous, yellow-brown, amber liquid. Platelet counts are in the range of approximately 1,000,000,000 to about 2,500,000,000 per ml and above, a 9- to 12-fold increase in the concentration in the collected blood. White blood cell values are from 30,000,000 to about 60,000,000 per ml, 6- to 9-fold of donor concentrations, with a composition of about 60% to about 70% lymphocytes, about 15% to about 25% monocytes, and about 5-25% neutrophils. Other white blood cells, e.g., basophils and eosinophils, are essentially absent. In particular, basophils and eosinophils are typically not present in detectable amounts. The lymphocyte concentration is increased by about 12- to about 15-fold, and monocytes are increased to about 20- to about 25-fold of the starting blood. Plasma protein concentrations are about 12 to about 20 g/dL. Fibrinogen concentrations are similarly about 2- to 3-fold higher than donor blood levels, as are other clotting proteins. Electrolyte concentrations are essentially unchanged from collected blood levels because electrolytes are passed through the membrane of the hemoconcentration device along with water and other small molecules.

The pore size, or sieving coefficient, of the membrane of the hemoconcentrator device is selected to retain proteins and other desired blood components, including any platelet components that may be released into the plasma during hemoconcentration. Ideally, the pore size, or sieving coefficient, corresponds to a 100% cutoff at 45,000 daltons, meaning that molecules larger than 45,000 daltons are retained in the plasma buffy coat concentrate.

After preparation, plasma buffy coat concentrate can be used immediately, or it can be stored, preferably at room temperature for best preservation of platelet function. After approximately 36 hours from the time of the original blood draw, deterioration in efficacy begins to set in, and after 5 days at room temperature it should be discarded.

Platelet concentrations of about 1,000,000,000 per ml are required to provide a clinically significant acceleration of wound healing when the plasma-buffy coat concentrate is used as a wound healing agent, and for clinically distinguishable adhesion enhancement when used as a wound sealant. If platelet counting has been performed prior to hemoconcentration with the plasma-buffy coat mixture and has shown the count to be greater than about 500,000,000 per ml, then platelet-poor plasma may be combined with the plasma-buffy coat mixture to a volume that brings the platelet count to about 500,000,000 per ml. Plasma and plasma-buffy coat mixture may be combined prior to hemoconcentration. Alternatively, the plasma-buffy coat mixture may be hemoconcentrated first and the plasma aliquot thereafter, with residual buffy coat components being flushed from the hemoconcentration device. By either method, a larger volume of plasma-buffy coat concentrate may be produced without a reduction in clinical efficacy.

In one embodiment for processing large volumes of blood to prepare the wound sealant, the method was carried out as follows. The method can be easily modified by one skilled in the art.

During assembly of a centrifugal blood fractionation kit (also referred to as a standard autotransfusion processing kit), a 1/4" isosceles three-way connector was placed with two of its arms between the cuff of the blood pump and the centrifuge basket, using a piece of 1/4" PVC tubing (conveniently about 15 cm) to connect the three-way connector to the centrifuge basket. The third arm of the three-way connector is in turn connected to a second piece of 1/4" PVC tubing (conveniently about 15 cm) which is attached to the inlet of a hemoconcentrator. The outlet of the hemoconcentrator is attached to a piece of 1/4" PVC tubing (about 2 to about 3 cm in length) which is in turn connected to the inlet coupler of a blood transfer bag. The tubing leading from the three-way connector to the hemoconcentrator is clamped.

Processing then proceeds by fractionating the blood into three components, packed red blood cells, plasma, and plasma-buffy coat mixture. After fractionation is complete, the packed red blood cells alone are pumped into the holding bag, being replaced in the centrifuge basket by sterile air previously present in one of the collection bags.

The plasma and plasma-buffy coat collection bags are then punctured with the two clamped autotransfusion processing kit piercing tubes that are usually reserved for wash solutions. The wash tube and fill tube are then swapped in the autotransfusion processing device clamp fitting so that a fill command draws fluid through the wash tube. The plasma collection bag piercing tube clamp is removed and the blood pump is started at approximately 50 mL/minute or less to draw plasma from the collection bag through the processing kit tubing and into the centrifuge bead, flushing any remaining blood cells from the tubing set into the centrifuge basket.

When the tubing is clear of red blood cells, the piercing tubing to the plasma bag is clamped and the piercing tubing to the plasma-buffy coat mixture collection bag is opened, drawing the plasma-buffy coat mixture toward the blood pump, with an air bubble separating the plasma-buffy coat mixture from the plasma. Once the air bubble reaches the three-way, the clamp on the tubing to the hemoconcentrator is removed and the tubing to the centrifuge basket is clamped. Flow now goes to the hemoconcentrator and a vacuum of approximately -400 torr is applied. When the plasma-buffy coat mixture reaches the hemoconcentrator, water is removed and the plasma-buffy coat concentrate flows into the transfer bag.

When all the plasma-buffy coat mixture has been emptied from its collection bag, the blood pump is temporarily stopped. The piercing tube of the plasma-buffy coat mixture collection bag is clamped, removed from the plasma-buffy coat mixture bag, and inserted into the transfer bag containing the buffy coat/plasma concentrate. If the water removal step did not remove the desired amount of water, the plasma-buffy coat concentrate may now be passed through the hemoconcentration device again until a sufficient amount of water has been removed. When removal of water is complete, the vacuum should be released from the hemoconcentration device. Recovery of residual plasma-buffy coat concentrate from the hemoconcentration device may be effected by pumping plasma from the plasma holding bag into the hemoconcentration device in a volume sufficient to displace the plasma-buffy coat concentrate into the transfer bag. As described above, other physiological solutions may also be used, but are less preferred since such solutions do not contain blood clotting proteins.

Mixing with a fibrinogen activator

Immediately before application to a wound, the plasma-buffy coat concentrate is mixed with a fibrinogen activator. The addition of the fibrinogen activator starts the clotting process, as described in detail below.

Fibrinogen activators are well known and commercially available, and include thrombin and batroxobin. Thrombin is preferred. Thrombin cleaves fibrinogen to fibrin monomer, which in turn cross-links to form a fibrin network.

Mixing a fibrinogen activator with the plasma buffy coat concentrate is a rapid and effective means of forming a sticky clot. Furthermore, thrombin is a potent activator of platelets, stimulating stickiness and initiating the release reaction in which the contents of platelet granules are released. Thrombin is commercially available from several sources, including Armour, Park-Davis, and others. Currently commercially available thrombin preparations are derived from bovine or equine sources. It Standard precautions should be taken for those with allergies or previous reactions to thrombin.

Unlike other fibrinogen-based sealant adhesives, the activated plasma buffy coat concentrate forms a coherent, strong adhesive after treatment with thrombin alone, without the need for the addition of calcium salts. Calcium present in the platelets has been found to be sufficient to enable effective cross-linking of fibrin. The presence of added calcium ions in the presence of platelets is generally undesirable because it allows the platelets to contract and expel serum from the clot, an undesirable transformation. Therefore, the fibrinogen activator alone in solution is preferred and is unexpectedly effective. However, in applications where slow clot formation is desired, calcium alone can be used to generate thrombin from the prothrombin present in the plasma buffy coat concentrate to form a clot over a period of several minutes.

To produce a coherent, uniform clot, the fibrinogen activator and plasma buffy coat concentrate must be uniformly mixed prior to contact with the target tissue surface. This is most conveniently accomplished by solubilizing the fibrinogen activator and then mixing the two liquids. Commercial thrombin preparations are provided in dry form with sterile saline as a diluent for reconstitution of the preparation. Other physiological solutions consistent with the preservation of thrombin activity and the components of the plasma buffy coat concentrate may be used. Clotting rates are adjusted to be sufficiently slow to permit application of the wound sealant, but fast enough to ensure rapid adhesion and sealing after application. In the presence of sufficient fibrinogen to form a clot, the rate of clot formation is directly dependent on the fibrinogen activator concentration in the final mixture. For example, at 100 U thrombin per ml, a clot forms in about 3 to 5 seconds, at 50 U/ml, the rate is about 15 to about 20 seconds, and at 10 U/ml, the clot forms in about 60 to about 120 seconds. Thus, the need of a particular application for faster or slower setting times (up to about one to two minutes) can be met by adjusting the fibrinogen activator concentration.

Temperature also affects the rate of clot formation. Applying small amounts, i.e. a few milliliters at a time in a thin layer, allows the target tissue to be heated sufficiently to maximally accelerate clot formation, without the need to warm the components to above room temperature before mixing. Higher temperatures, up to 42ºC, may be used, although such temperatures offer no advantage. If desired, lower temperatures can be used to prolong the clotting time.

Volume ratios of the two fluids are important in determining the strength, stickiness, and platelet concentration of the wound sealant. Minimizing dilution of the fibrinogen and platelets by the fibrinogen activator solution maximizes these concentrations. Although volume ratios of plasma buffy coat concentrate to fibrinogen activator solution of about 5:1 have been used successfully, the frequency of unsatisfactory results is higher than with higher ratios. Ratios of about 10:1 to about 12:1 are reliably effective. Higher ratios make thorough mixing of the two solutions more difficult and unreliable.

To achieve the desired degree of uniform mixing, two methods were used. Both methods are preferably carried out at room temperature. In the syringe method, about 10 to about 12 ml of plasma buffy coat concentrate is drawn into a syringe, along with about 1 or 2 ml of air to facilitate mixing. One ml of fibrinogen activator solution is then drawn in and the syringe is rotated about 180 degrees vertically about three times, allowing an air bubble to rise along the length of the syringe and thus repeatedly mixing the two solutions.

Alternatively, the two solutions can be mixed in a small cup or container using a stick, spatula or other suitable instrument by rapidly stirring them together. Certain commercially available mixing devices, such as dual fluid flush systems (available from Research Medical and Hemaedics) have been tested but found to be unsuitable. Such devices are designed for a 1:1 volume mixing ratio, causing undesirable dilution, and cannot be easily adapted to higher volume ratios; and clotting with resulting clogging frequently occurred in the Hemaedics device. For maximum adhesion to tissue surfaces, application of the wound sealant must occur during gelation. With the syringe method, the wound sealant can be applied directly to the desired site using a cannula, catheter, endoscope, or other suitable tube or nozzle as appropriate for the situation. Most effectively, the wound sealant is applied in a thin layer, with additional layers as needed for additional strength.

The coagulating wound sealant can be poured from a cup onto the desired site while it is still liquid, or spread with a spatula or similar tool. In either case, a relatively dry site offers the best opportunity for effectiveness. Holding a sponge or pad over the intended site until the time the wound sealant is applied is very beneficial.

Blood processing kits

The present invention also provides a blood processing kit to form a plasma buffy coat concentrate. The kit comprises a blood fractionation kit by centrifugation and a hemoconcentration device. As described above, a blood fractionation kit by centrifugation comprises a centrifuge basket, tubing and collection bags for removing red blood cells using a dual centrifugation technique. Preferably, the components of the kit are sterile and for single use. According to another embodiment, the kit also comprises blood bag spikes, stopcocks and tubing for preparing the plasma-buffy coat concentrate.

It is understood that application of the teachings of the present invention to a specific problem or situation is within the skill of one of ordinary skill in the art having knowledge of the teachings contained herein. Examples of the products of the present invention and representative methods for their isolation, use and preparation are provided below, but should not be construed as limiting the invention. All literature citations herein are expressly incorporated by reference.

EXAMPLE 1

In an exemplary preparation and use of the wound sealant of the present invention, a 57-year-old obese diabetic woman presented with an unhealed wound and a persistent large cerebrospinal fluid (CSF) leak of unknown origin six weeks after multiple vertebral laminectomy. Three revision surgeries failed to identify the site of the leak, and attempts to seal the leak with a blood patch, collagen foam, grafted fat pads, compression, and bed rest had all failed. Systemic and topical antibiotic irrigation, repeated tissue debridement, wet and dry packing, and placement of drainage had failed to manage the unhealed wound, which was now 50% larger than the original surgical incision.

After induction of anesthesia for her fourth procedure, one unit of her own blood was sterilely drawn from the anterior antecubital vein. The blood was collected in a standard sterile blood collection set using the anticoagulant CPDA-1 (Terumo Corporation). Analysis of the collected blood showed a hematocrit of 34% and a platelet count of 224,000,000. The white cell count was 7,200 per mL, with a differential analysis showing 63% neutrophils, 29% lymphocytes, 5% monocytes, 2.2% eosinophils, and 0.8% basophils. Fibrinogen was tested at 2.85 mg/mL.

The collected blood was connected to a sterile disposable blood processing set, manufactured by Electromedics, Inc., which in turn was connected to an ELMD500 autotransfusion system, also manufactured by Electromedics. The centrifuge basket (125 mL) was set to a rotation speed of 5600 rpm, which creates forces on the order of 2000 G in the blood path of the basket. The blood was then pumped into the basket at 50 mL per minute.

Once the basket was almost completely filled with packed blood cells and only a small central core of clear plasma remained, the blood pump was stopped and the centrifuge speed was reduced to 2400 rpm. The outlet tube leading to the plasma collection bag was closed, the outlet tube leading to another collection bag was opened, and blood flow was restored at 50 ml/min. The blood now flowing into the centrifuge was again fractionated, but at the lower centrifugal force, leaving the red blood cells and neutrophils in the centrifuge basket.

The plasma fraction continued to flow out of the basket, carrying most of the platelets, monocytes and lymphocytes into the second collection bag, forming a plasma-buffy coat mixture. Blood continued to be pumped into the basket so that it filled with red blood cells, forcing the central plasma column and entrained entities into the outlet tube. Flow was continued until red blood cells began to flow into the second collection bag, at which time blood flow was stopped, the outlet tube leading to the bag containing the plasma-buffy coat mixture was clamped, and the centrifuge was stopped.

The plasma collection bag containing the plasma-buffy coat mixture was then opened. The direction of the blood pump was reversed. Using clamps, the platelet-poor red blood cells and plasma were transferred to a third collection bag. The procedure was repeated until all of the blood that had been collected had been processed. The reconstituted, platelet-depleted whole blood was then returned to the patient.

The elapsed processing time for the two-speed centrifugation fractionation procedure was 18 minutes. The yield of plasma-buffy coat mixture was 45 mL, with the platelet count being 628,000,000 per milliliter and the white blood cell count being 14,300 per milliliter. The leukocyte differential analysis showed 72% lymphocytes, 21% monocytes, 6% neutrophils, and less than 1% other types. The hematocrit was 0.3. Fibrinogen was 2.80 mg/mL. This plasma-buffy coat mixture was subsequently processed.

A sterile construction of commercially available blood bag spikes, stopcocks and tubing was assembled such that the bag of platelets was connected to a three-way stopcock. A second arm of the stopcock led to a 60 cc syringe and the third arm to a hemoconcentrator manufactured by Minntech, Inc. with a surface area of 0.25 square meters, the outlet of which was in turn connected to a sterile blood transfer bag from Terumo Corporation. The plasma-buffy coat mixture was then drawn into the 60 cc syringe. By adjusting the connected stopcock, the flow path between the syringe and the hemoconcentrator was opened. A vacuum of -400 Torr was applied to the discharge port of the hemoconcentrator and the plunger of the 60 cc syringe was actuated to force plasma through the blood path of the hemoconcentrator for approximately two minutes, producing a plasma-buffy coat concentrate.

The vacuum was immediately released and the empty 60 cc syringe was replaced with a 20 cc syringe filled with physiological saline. The saline was immediately injected into the hemoconcentration device to flush residual plasma-buffy coat concentrate from the device into the receiving transfer bag, forming the final plasma-buffy coat concentrate. This curtain took only one minute to complete.

The plasma buffy coat concentrate was 18 ml of a viscous, yellow-brown, amber liquid. Analysis showed a platelet count of 1,532,000,000 and a white blood cell count of 35,600 per milliliter, with unchanged cell type composition. Fibrinogen was measured at 6.68 mg/ml and the hematocrit was measured at 0.5%.

This plasma-buffy coat concentrate was drawn into a 20 cc syringe and transferred sterilely into a medication cup in the surgical sterile field. Nine ml of the plasma-buffy coat concentrate was drawn into two 12 cc syringes together with 2 ml of air, which were then fitted with 14 gauge plastic needles and set aside. In addition, 5,000 units of bovine thrombin USP (Thrombostat, from Parke-Davis) were dissolved in 5 ml of accompanying sterile saline to give a concentration of 1,000 units per milliliter.

After the surgeon had completed debridement and further treatment of the wound and revealed an obvious site for a CSF leak, the surgical assistant took one of the 12 cc syringes and aspirated one ml of thrombin solution. By rotating the syringe 180 degrees vertically, the air bubble caused rapid mixing of the two fluids. The syringe was given to the surgeon, who immediately applied the contents to the desired tissue site. Within 5 seconds, a whitish clot formed covering the site of the suspected CSF leak. For additional stability, a piece of autologous muscle skin was sutured over the clot. The contents of the second syringe of plasma buffy coat concentrate were treated similarly and applied in a thin layer to the remaining wound surfaces. The wound was closed in the conventional manner, with a small silicone sump drain and a dry skin dressing.

Forty-eight hours after surgery, the patient was raised to a sitting position, apparently without CSF leak. The wound drain was less than 10 cc, and 72 hours after surgery, the drain was removed. The wound showed conventional healing with minimal redness of the surrounding tissue and no wound exudate. Four days after surgery, the patient was discharged uneventfully. At a follow-up examination at three months, the patient showed a completely healed wound with no evidence of CSF leak.

EXAMPLE 2

In another exemplary use of the inventive platelet adhesive wound sealant, a 72-year-old, 58 kg female was presented for open heart surgery and cardiopulmonary bypass with replacement of her ten-year-old mitral valve prosthesis and triple coronary artery bypass graft. Research and experience have shown that this type of patient has significant risk factors for blood loss and transfusion requirements, including advanced age, low body mass, reoperation, and combined cardiac surgery. This patient would be expected to produce 1,500 ml or more of postoperative chest wound drainage and is at 50% or greater risk of requiring transfusion, with a 30% or greater risk of multiple transfusions.

After induction of anesthesia, blood was drawn through a catheter placed in the right internal jugular vein using an Electromedics double line connected to the inlet line of the disposable blood processing set. Anticoagulant citrate solution, also from Electromedics (3 mg citric acid (anhydrous) USP, 26.3 mg sodium citrate (hydrated) USP, sodium dihydrogen phosphate (monohydrate) USP, and 23.2 mg dextrose USP) was connected to the second line and thereby mixed with the patient's blood during collection, in a ratio of 15 mL citrate solution to 100 mL patient blood.

An inlet tube from the disposable blood processing set manufactured by Sorin, Inc. was connected to an autotransfusion system blood pump (Dideco Inc., Model BT795AT), which controlled the flow of blood from the patient. For the first separation step, the centrifuge speed was set at 4800 rpm and the blood flow was started at 100 ml/minute. The centrifuge basket (225 ml capacity) was filled as described in Example 1 before generating a cell-poor plasma fraction. As in Example 1, the blood flow was stopped, the centrifuge speed was reduced to 2400 rpm, and the Blood flow was resumed at 50 ml/min, producing a buffy coat fraction with a volume of 68 ml.

After collection into a syringe, the fractionation procedure was continued to obtain a supply of autologous plasma-buffy coat mixture and plasma for reinfusion after interruption of cardiopulmonary bypass, while red blood cells were returned to the patient before the start of bypass surgery.

The plasma-buffy coat mixture was concentrated as in Example 1, this time using a hemoconcentrator from Amicon Inc. The volume of plasma-buffy coat concentrate produced was 26 ml. The analysis of the blood in its various processing steps (after collection from the patient, in the plasma-buffy coat mixture and in the plasma-buffy coat concentrate) was as follows:

After completion of cardiopulmonary bypass, the remaining complexed plasma and plasma-buffy coat concentrate not used for wound sealing were administered intravenously. The plasma-buffy coat concentrate was brought to the sterile field as described in Example 1, together with 10,000 units of Thrombin USP (Armour, Inc.) dissolved in 10 cc of physiological saline. The plasma-buffy coat concentrate was similarly aspirated into two 20 cc syringes, and the Thrombin solution was similarly transferred into two 5 cc syringes. Each syringe was fitted with a spray nozzle tip from Hemaedics, Inc.

When application was desired, the surgeon held a syringe of plasma buffy coat concentrate in one hand and a syringe of thrombin in the other, applying their contents to the tissue surfaces in an overlapping spray pattern. The contents of all four syringes were applied, covering the mediastinum, the resected adhesions covering the cardiac surface, the cardiotomy sites, the aortic root, the internal chest walls, and the cut sternum. Drains were placed and the wounds were closed according to a standard protocol.

The total postoperative chest mediastinal drainage was 358 ml, and on the morning after surgery the patient was no longer ventilated and transferred from the intensive care unit to a nursing unit. The postoperative course was uneventful and the patient was discharged on day 6 without the need for allogeneic blood transfusion.

EXAMPLE 3

The effect of reactivation of endogenous thrombin in plasma buffy coat concentrate on gel time was investigated. The table below illustrates the effect of various ratios of 10% calcium citrate to plasma buffy coat concentrate at 22ºC on gel time. No exogenous fibrinogen activator was used in this study.

Volume ratio of 10% CaCl2 to plasma buffy coat concentrate / gel time at 22ºC

1 : 5 30

1 : 10 12

1 : 11 11

1 : 12 11

A study was conducted to investigate the effect of temperature on gel time. In the study, one part of 10% CaCl2 solution was mixed with 11.5 parts of plasma buffy coat concentrate.

Temperature / gel time

22ºC 11 minutes

32ºC 5 minutes

37ºC 2.5-3 minutes

As can be seen from this study, gel times of two to thirty minutes can be produced, if desired, by adding calcium ions to the plasma buffy coat concentrate.

EXAMPLE 4

This example describes studies using a plasma buffy coat concentrate and dry bone meal, with clot formation induced by treatment with calcium chloride. The study was performed using a plasma buffy coat concentrate prepared from bovine blood with a fibrinogen concentration of 570 mg/dL and a platelet count of 1,200,000,000/mL.

Six different compositions were studied. The bovine plasma buffy coat concentrate was used alone or combined with either 10% CaCl2 or a commercial preparation of ground dry bone meal. The results of the study are shown in the table below. In the table, the ratio of plasma buffy coat concentrate to CaCl2 is in ml concentrate: ml CaCl2, and the ratio of concentrate to bone meal is in ml concentrate: cc bone meal. Thrombin was not added to any of the compositions. All compositions were covered and left to stand at room temperature (22ºC).

* Not applicable: indicates that CaCl₂ or bone meal was not present.

By comparing samples (2) and (3) with (1), this study demonstrated that calcium ions alone are sufficient to clot plasma buffy coat concentrate, albeit slowly, when present in excess of the amount required to neutralize all free citrate. Samples (4), (5) and (6) demonstrated that dry bone meal, predominantly hydroxyapatite, can have the same effect. This may occur due to complexation of the citrate by the solid bone, thereby releasing calcium ions for clot formation, or by dissolving calcium ions from the bone matrix, providing sufficient free calcium ions to allow clotting to occur, or by a combination of these. Sample (4) also demonstrated that only a small amount of bone meal is required to clot a significantly larger volume of plasma buffy coat concentrate. Samples (5) and (6) showed that by mixing increasing amounts of bone meal relative to the volume of plasma buffy coat concentrate, increasingly stronger gels could be produced. These gels immediately after mixing exhibit good working properties that would allow the formation of any desired shape or configuration. When left to stand, these mixtures attain greater hardness and strength.

An advantage of using calcium over other techniques for initiating coagulation is the absence of the need for exogenous thrombin, a source of a certain number of complications in patients. Another advantage demonstrated by samples (4), (5) and (6) is that the composition can be formed into virtually any desired configuration, and particularly in samples (5) and (6), the formation of a solid paste eliminates the tendency of the sealant to run and move away from the intended application site prior to clot formation. At the same time, the plasma buffy coat concentrate converts the bone meal from a fine particulate consistency to a workable paste, facilitating application to any recipient site. Another advantage is the presence of tissue growth stimulating substances from the platelets, which when used with autologous bone mass have been shown to accelerate and complete wound healing. The same advantage could be applicable to hydroxyapatite or other exogenous source of bone growth matrix without the need for thrombin treatment.

This study illustrates the benefits of using the platelet adhesive wound sealant for bone repair in humans and in similar veterinary applications.

Claims (22)

1. Plasma buffy coat concentrate comprising: a. Plasma, b. platelets at a concentration of at least 1.0 x 10⁹ cells/ml, c. Fibrinogen at a concentration of at least 5 mg/ml, and d. white blood cells at a concentration of at least 3.0 x 10⁷ cells/ml. 2. The plasma buffy coat concentrate of claim 1, wherein the platelet concentration is from about 1.0 x 109 cells/ml to about 2.5 x 109 cells/ml. 3. The plasma buffy coat concentrate of claim 1, wherein the fibrinogen concentration is from about 5 to about 15 mg/ml. 4. The plasma buffy coat concentrate of claim 1, wherein the white blood cell concentration is from about 3.0 x 107 cells/ml to about 6.0 x 107 cells/ml. 5. The plasma buffy coat concentrate of claim 4, wherein the white blood cells comprise about 60 to about 70% lymphocytes, about 15 to about 25% monocytes, and about 5 to about 25% neutrophils. 6. A wound sealant comprising: a. Plasma, b. platelets at a concentration of at least 1.0 x 10⁹ cells/ml, c. Fibrinogen at a concentration of at least 5 mg/ml, d. white blood cells at a concentration of at least 3.0 x 10⁷ cells/ml, and e. a fibrinogen activator in a concentration sufficient to initiate clot formation. 7. The wound sealant of claim 6, wherein the fibrinogen activator is selected from the group consisting of thrombin and batroxobin. 8. A wound sealant according to claim 7, wherein the fibrinogen activator is thrombin. 9. The wound sealant of claim 8, wherein the thrombin is generated from prothrombin present in the plasma component of the wound sealant. 10. A method for processing blood, comprising: a. Fractionating anticoagulated blood by centrifugation to remove red blood cells and produce a plasma-buffy coat mixture, and b. hemoconcentrating the plasma-buffy coat mixture to remove water from the mixture, thereby producing a plasma-buffy coat concentrate according to claim 1. 11. The method of claim 10, further comprising mixing the plasma buffy coat concentrate with a fibrinogen activator to produce a wound sealant. 12. The method of claim 10, wherein the blood is processed perioperatively. 13. The method of claim 10, wherein the volume of water removed from the plasma in step (b) is between about 50 and about 75 percent of the original plasma volume. 14. The method of claim 10, wherein the leukocyte concentration of the plasma-buffy coat concentrate is at least five times the leukocyte concentration of the blood. 15. The method of claim 14, wherein the concentrations of lymphocytes, monocytes and platelets of the plasma-buffy coat concentrate are at least ten times the corresponding concentrations of the blood. 16. A method for producing a wound sealant according to claim 6, comprising: a. Fractionation of anticoagulated blood by centrifugation, whereby red blood cells are removed and a plasma-buffy coat mixture is produced, b. hemoconcentrating the plasma-buffy coat mixture to remove water from the mixture, thereby producing a plasma-buffy coat concentrate, and c. Mixing the plasma buffy coat concentrate with a fibrinogen activator to produce a wound sealant. 17. The method of claim 16, wherein the blood is human blood. 18. The method of claim 16, wherein the blood is from the same animal to which the wound sealant is to be applied. 19. A kit adapted to process blood according to the method of claim 10, comprising a set for fractionating blood by centrifugation and a hemoconcentration device. 20. The blood processing kit of claim 19, wherein the kit additionally comprises a blood bag spike, a stopcock and tubing. 21. The blood processing kit of claim 20, wherein the components of the kit are disposable components. 22. Use of a plasma buffy coat concentrate according to claim 1 in the manufacture of a sealant to promote the sealing of a wound.